Structural Analysis of the Human Rad51 Protein-DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis

Transmission of Allosteric Effects between ATP Binding and DNA Binding

Axelle Renodon-Cornière, Yoshimasa Takizawa, Sébastien Conilleau, Vinh Tran, Shigenori Iwai, Hitoshi Kurumizaka, Masayuki Takahashi

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    Abstract

    Abstract: Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.

    Original languageEnglish
    Pages (from-to)575-587
    Number of pages13
    JournalJournal of Molecular Biology
    Volume383
    Issue number3
    DOIs
    Publication statusPublished - 2008 Nov 14

    Fingerprint

    Tryptophan
    Adenosine Triphosphate
    Fluorescence
    DNA
    Proteins
    Binding Sites
    Adenosine Triphosphatases
    Molecular Models
    Homologous Recombination
    Phenylalanine
    Fluorescent Dyes
    Histidine
    Alanine

    Keywords

    • allosteric effect
    • homologous recombination
    • protein filament
    • Rad51 protein
    • tryptophan fluorescence

    ASJC Scopus subject areas

    • Molecular Biology

    Cite this

    Structural Analysis of the Human Rad51 Protein-DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis : Transmission of Allosteric Effects between ATP Binding and DNA Binding. / Renodon-Cornière, Axelle; Takizawa, Yoshimasa; Conilleau, Sébastien; Tran, Vinh; Iwai, Shigenori; Kurumizaka, Hitoshi; Takahashi, Masayuki.

    In: Journal of Molecular Biology, Vol. 383, No. 3, 14.11.2008, p. 575-587.

    Research output: Contribution to journalArticle

    Renodon-Cornière, Axelle ; Takizawa, Yoshimasa ; Conilleau, Sébastien ; Tran, Vinh ; Iwai, Shigenori ; Kurumizaka, Hitoshi ; Takahashi, Masayuki. / Structural Analysis of the Human Rad51 Protein-DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis : Transmission of Allosteric Effects between ATP Binding and DNA Binding. In: Journal of Molecular Biology. 2008 ; Vol. 383, No. 3. pp. 575-587.
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    abstract = "Abstract: Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.",
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    T2 - Transmission of Allosteric Effects between ATP Binding and DNA Binding

    AU - Renodon-Cornière, Axelle

    AU - Takizawa, Yoshimasa

    AU - Conilleau, Sébastien

    AU - Tran, Vinh

    AU - Iwai, Shigenori

    AU - Kurumizaka, Hitoshi

    AU - Takahashi, Masayuki

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    N2 - Abstract: Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.

    AB - Abstract: Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.

    KW - allosteric effect

    KW - homologous recombination

    KW - protein filament

    KW - Rad51 protein

    KW - tryptophan fluorescence

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